The long term objective of this proposal is to understand the mechanisms by which herpes simplex virus (HSV) cleaves and packages genomic length viral DNA into preformed capsids. Proteins required for packaging HSV DNA would be expected to specifically bind and cleave DNA Pac sequences to form genomic termini and hydrolyze ATP for translocating DNA. The HSV gene products likely to implement these steps include the Ul15 and Ul36 encoded proteins. Data indicate that the Ul15 gene is required for DNA cleavage packaging, and encodes two proteins of apparent M 35,000 and 75,000. The HSV-1 U136 encoded protein VP1 because it has been shown to specifically bind the HSV-1 Pac sequences. The specific objectives are: 1. Determine the origin of the proteins encoded by UL15. To distinguish the possibilities that the 35,000-M, protein is expressed by proteolytic cleavage, a novel promoter, or alternative splicing we will (i) determine the N-terminal amino acid sequence of the 35,000 m protein or (ii) perform immunologic analyses of viruses containing epitopically tagged UL15 genes. 2. Determine the role of the UL15 gene products in DNA cleavage and packaging. Examination of DNA cleavage and packaging in cells infected with viral mutants expressing null genes and single UL15 proteins will indicate the function of each gene product. Both proteins and individual UL15 proteins expressed in heterologous systems will be tested for ATP and DNA binding. Introduction of genes bearing mutations ablating these activities into the viral genome and examination of DNA cleavage and packaging in cells infected with these viral mutants will indicate the role of U115 protein DNA binding and ATPase activities in viral DNA cleavage and packaging. 3. Determine the role of other proteins in DNA packaging. VPI proteins expressed in heterologous systems will be tested for DNA binding and ATPase activities. If the protein is not capable of binding Pac site DNA specifically in the absence of other proteins, steps will be taken to identify the gene expressing a 140,000 M, protein previously shown to copurify with VP. Mutant VP1 genes will be expressed in heterologous systems and will be tested for DNA binding and ATPase activities to identify sequences required for these activities. Analysis of cells infected with viral mutants expressing null and mutant VPI proteins not able to express these activities will indicate the function of VPI and VP1- associated activities in DNA cleavage and packaging. Studies are also proposed to identify and characterize the UL6. UL25, and UL33 encoded proteins in infected cells and test the respective proteins for DNA binding and ATPase activities.